Abstract Smoking is the foremost preventable cause of death. Nicotine (NIC) determines the addictive nature of smoking. In rats, NIC addition has been commonly modeled by intravenous NIC self-administration (SA). We (Dr. Vezina) have recently shown that NIC sensitization, a process that appears after repeated exposure to NIC, can enhance the SA of NIC and other stimulants. NIC sensitization and SA in rats are known to correlate with neurobiological changes in multiple brain regions, however, the molecular basis of the neurobiological changes of NIC sensitization and SA as well as its relevance to human NIC abuse has yet to be determined. We propose to take advantage of the depth and breadth of RNA sequencing (RNAseq) to identify the gene expression profiles of NIC sensitization and SA by quantifying transcripts actively involved in protein synthesis (i.e., translatome) in these processes, and to explore its relevance to enhanced NIC use in humans. Translatome profiling better predicts protein abundance than conventional transcriptome profiling and has higher resolution and dynamic range than mass spectrometry-based proteomic assays. To model NIC sensitization and SA, we will innovatively use F1 progeny of two inbred strains, Fischer-344 (F344) and Brown Norway (BN) since F1 rats are (1) genetically identical, thus minimizing ?genetic noise? on measured differential expression and (2) heterozygous at all loci where parental strains have different alleles, thus maximizing the informative heterozygous variants for analyzing allele-specific expression (ASE). We have shown that these F1 rats (F344/BN) exhibit robust NIC sensitization. AIM1: We will characterize the translatome profiles of NIC sensitization and SA by sequencing RNA transcripts bound to ribosomes in ventral tegmental area (VTA) and nucleus accumbens (NAc), the brain regions most relevant to NIC addiction. We will further identify genetic variants showing ASE of sensitization-associated gene translation. AIM2: To examine the relevance of NIC addiction-associated translatome profiles to the enhanced NIC use in humans, we will derive induced pluripotent stem cells (iPSCs) and midbrain DA neurons from both smokers and non-smokers, expose DA neurons to NIC, and examine how the NIC-induced expression changes in iPSC-neurons correlate with those observed in rat brain tissues. For genes correlated with NIC sensitization, we will further evaluate their relevance to human smoking behavior by performing a pathway-based association test in publicly available smoking GWAS datasets. Identifying novel gene targets relevant to NIC sensitization and SA will forge a new path to deepen our understanding of the neurobiology of human NIC abuse, helping develop more effective therapeutic interventions.